Correlative Light and Electron Microscopy: Methods and Applications


Light has its limits, and even in the world of ‘super resolution’ microscopy, many cellular structures and even large protein complexes are not resolvable in the light microscope – although it is ideal for watching living cells and tissues and linking structural changes to functional outcomes. Electron microscopy can yield molecular level resolution but is limited to nonliving samples that have been extensively processed to preserve structure and be visible using electron optics. While it would be ideal to have a single system which provided nanoscale information on living samples, until that day arrives, the obvious answer is to use both light microscopy and electron microscopy as complementary approaches. Localising the same labelled structure in both imaging modalities is known as correlative LM and EM or correlated light and electron microscopy. Various methods and instrumentation are used for correlating data from the same sample imaged using both light and electrons.

Key Concepts

  • Light microscopes cannot resolve specimens at the nanoscale.
  • Combining light and electron microscopy can link ultrastructure to function.
  • For CLEM, exactly the same region of interest (ROI) is imaged in both the light and electron microscope.
  • 3D (volume) electron microscopy can provide spatial information at the nanoscale.
  • Using special probes, proteins can be located in both light and electron microscopes.

Keywords: correlative light and electron microscopy; light microscopy; electron microscopy; immunochemistry; horse radish peroxidase; fluorescence; photooxidation; APEX

Figure 1. In‐resin fluorescence. CLEM of GFP‐C1 and mCherry‐H2B in HeLa cells using postembedding light microscopy. GFP‐C1 fluorescent signals overlaid onto electron micrographs of the corresponding region using the IRF method. (a) Image of GFP‐C1 signal alone imaged under dry conditions, and overlaid directly onto the matching electron micrograph, for a cell embedded in HM20. The fluorescent signal corresponds to the nuclear envelope, and the other structures within the cytoplasm. (b) An adjacent section through the cell shown in (a). (c–e) Increased precision of GFP‐C1 localisation to Golgi stacks (g) and the NE. In (c), black arrows indicate localisation of fluorescent signal to the highly curved tips of Golgi cisternae. An enlarged view from an adjacent section is shown in (d). Localisation to the NE is also evident. In (e), white arrows indicate localisation of fluorescent signal to the nucleoplasmic reticulum. (f, g) Localisation of GFP‐C1 to endoplasmic reticulum and to membrane stacks (black arrows) in cells expressing low (f) and high (g) levels of the GFP‐C1 construct. ER, endoplasmic reticulum; G, Golgi; M, mitochondrion; N, nucleus; NE, nuclear envelope. Scale bars – (a, b): 5 µm, (c, d): 500 nm, (e–g): 1 µm. Reprinted by permission from 2014 © Elsevier.
Figure 2. Overview of CLEM labels. Several CLEM labels have been developed over the years. Here, we have composed an overview of the labels discussed and have divided them into two categories: immuno‐ and genetic labels. Any label in this overview has successfully been used for CLEM experiments, but each has its advantages and disadvantages. Which label can be used varies for each project and depends on the biological question and feasibility. In general, a label that ensures optimal imaging in both LM and EM should be chosen.
Figure 3. Double‐labelled probes for visualisation in LM and EM. (a) Stills from a movie taken after 5 min of internalisation of both Tf–Alexa594–5 nm Au and EGF–Alexa488–10 nm Au (time indicated in seconds). The arrow points to a structure where the probes are initially colocalised. At time point 0, there appears to be segregation between the red fluorescence on the left and green on the right. At this point, the sample was taken and high‐pressure frozen and processed for EM. (b) To study whether the apparent segregation of the probes in the light microscope was true, the same cell was retraced in the EM. (c) An overlay of the last fluorescent frame onto this EM overview helps to identify the region of the cell where the structure of interest is located (boxed area in (b)). The structure of interest indeed shows segregation in the distribution of Tf–Alexa594–5 nm Au (red arrows) and EGF–Alexa488–10 nm Au (green arrows). Reprinted from Brown and Verkade, 2010 © US National Library of Medicine National Institutes of Health.
Figure 4. Photooxidation of Alexa dyes. (a, b) Endocytic uptake of Alexa 568‐labelled high‐density lipoproteins. HepG2 cells, on day 3 after seeding, were incubated with 50 µg mL–1 of high‐density lipoprotein Alexa 568 for 1 h. Cells, washed twice with PBS, were fixed and illuminated for 20 min utilising a TRITC filter. The intensely stained organelles in the light microscope (a) after photooxidation are identified as multivesiculated bodies at the EM level (b). (c, d) Chinese hamster ovary cells transiently transfected with ‐acetylgalactosaminyltransferase‐1 (GalNacT‐1). The cDNA sequence from GalNacT‐1 was cloned into the mammalian expression vector pEGFP‐N (Clontech Laboratories, Mountain View, CA, USA) with the GFP on the ‐terminus. Twenty‐four hours after transfection, the cells were fixed and illuminated for 30 min using a FITC filter setting. (c) Fluorescence light microscopy shows the Golgi resident GalNAcT‐1GFP localised to a juxta‐nuclear region. (d) After photooxidation, the DAB deposits are localised to cisternal lumina at one side of the Golgi stack. Reprinted by permission from Meisslitzer‐Ruppitsch et al., 2009 © John Wiley and Sons.
Figure 5. APEX, the GFP for EM? (a) EM images of HEK cells stably expressing MICU1‐APEX2. Two fields of view are shown. (b) Mitochondria of untransfected HEK cells processed under identical conditions. Scale bars–500 nm. Reprinted by permission from Lam et al., 2015 © Nature Publishing Group.
Figure 6. Using NIRB to approach and delineate ROI in 3D‐SEM. (a) Schematic illustrating the location of the ROI. (b) Maximum intensity projection of a confocal image stack in the ROI. (c) Fiducial branding marks applied to the ROI by NIRB and visualised by their autofluorescence. Arrows indicate horizontal and vertical marks delimiting the ROI, as burned into the tissue a few micrometers more ventral than the position of the neuron of interest. Two additional horizontal marks were applied adjacent to the DC ms axon branch point, ‘clasping’ it at the same ventral–dorsal depth (outlined in green). (d) The strategy for approaching and quickly finding the ROI in the resin‐embedded VNC. A guiding line is branded from the anterior end of the tissue to close to the ROI (long yellow line in magnified inset). This mark can be followed when cutting and imaging the neuropil from anterior to posterior in transverse direction (illustrated by grey plane and arrows). Additional smaller marks are placed to provide information about progress in the anterior–posterior directions (smaller vertical lines). (e) Branding marks (modelled in (d)) visualised by autofluorescence. (f) EM image of a transverse section during approach of the ROI, at the position corresponding to dotted lines in magnified (d) and (e). Branding marks are promptly identified in the tissue (arrowheads, numbers as in (d)). (g) Single confocal section in the plane of the neuronal branch point, clasped by two horizontal branding marks (arrows). (h) EM image of a transverse section in which the ms axon (pseudocoloured red) is clasped by the two horizontal branding marks (yellow). (h′) A magnification of the boxed area in (h), showing that the ultrastructure of cellular components, such as axonal microtubules (arrowheads) and synaptic vesicles (asterisk), is well preserved even in close proximity to the branding mark. (i) 3D reconstruction of the ms axon (red) and the branding marks (yellow) segmented from the stack of EM images in the ROI. Identity of the neuron is confirmed by correlating its morphology and relative position to the marks with the light microscopy data (c). Body axes are indicated in (b), (e–i); a, anterior; d, dorsal; p, proximal. Scale bars – (b): 10 µm, (e): 50 µm, (f): 2 µm, (g, h): 5 µm, (h′): 1 µm. Reprinted by permission from Urwyler et al. (2015). Open Access article distributed under the terms of the Creative Commons License Attribution 3.0 Unported.


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Further Reading

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Kremer, Anna, Lippens, Saskia, and Guérin, Christopher J(Jan 2016) Correlative Light and Electron Microscopy: Methods and Applications. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0025983]